Secretory as well as many membrane proteins are initially synthesized as nascent intracellular preproteins with a signal peptide attached to the N-terminus. The signal peptide enables the protein to cross the inner membrane barrier. In this process the protein gets cleaved and released as a mature protein which normally resides in the periplasmic space. Some exceptions to this mechanism are certain membrane proteins whose signal peptides remain uncleaved (S. Letenhardt, et al. in Protein Engineering, Application in Science, Medicine and Industry edt. by M, Inouye and R. Sharma, 1986 Academic Press, Inc., 157-171). A few prokaryotic proteins or peptides are synthesized as large precursors possessing signal peptides (pre-region) as well as pro-regions. Both segments are cleaved to yield mature proteins or peptides. Examples include subtilin of Bacillus subtilis (C. Nishio, et al, 1983, Biochem. Biophys. Res. Commun. 116, 751-758) or stable toxin of Escherichia coli (P. Dwarakanath, et al, 1989, Gene 81, 219-226). Enterotoxigenic E. coli (ETEC) strains cause diarrhoea in humans by the elaboration of extra cellular toxins classified as heat labile (LT) and heat stable (ST) family of toxins (R. N. Greenberg and R. L. Guerrant (1981). Pharmacol. Ther. II, 507-537; M. D. Gill and M. Woolkalis 1985 in Microbial toxins and diarrhoeal diseses, Ciba Foundation symposium 112, Pitman, London, 57-73). ST toxins are of two types: methanol soluble toxins (STI) and methanol insoluble toxins (STII). STI is further classified into 3 groups depending upon the origin i.e. STh (human), STp (porcine) and STb (bovine). The genes for both the LT and ST are plasmid encoded. The nucleotide sequence of the st gene is shown in FIG. 1. (P. Dwarakanath et al, 1989, Gene 81, 219-226). From the nucleotide sequence as well as from the translated amino acid sequence it is concluded that the 72 amino acid peptide is a precursor of ST which is processed post translationally to release 19 amino acid peptide from the carboxy terminal as the biologically active toxin. (P. Dwarakanath et al, 1989, Gene, 81, 219-226).
Attempts have been made by many groups to utilize the "signal sequence" portion of naturally occuring secretory proteins to construct recombinant vectors that will secrete heterologous proteins. In many cases synthetic signal sequences are also used. In all these cases the recombinant products are localized within the subcellular compartments. Specific examples are: A patent by Gray et al (U.S. Pat. No. 4,755,465 Jul. 5, 1988) where the inventors have claimed that they have constructed a vector which promotes the secretion of correctly processed human growth hormone (hGH) in E. coli and Pseudomonas. The "signal sequence" which also comes under the patent claim is very different from E. coli stable toxin "signal sequence".
II. A second patent (U.S. Pat. No. 4,704,362 dated Nov. 3, 1987) by Itakura et al. describes a recombinant cloning vehicle for microbial polypeptide expression where a fusion product of .beta.-gal and somatostatin is produced and then processed in vitro to get the final product.
An example where specifically an enterotoxin signal sequence is employed, is by Gray et al. (U.S. Pat. No. 4,680,262, 1985) where the inventors linked methanol insoluble stable toxin (STII) "signal sequence" with human growth hormone (hGH) gene and localized the product in the periplasmic region of the host cell. It is interesting to note that inventors were specifically looking for an expression vehicle which localized the expressed recombinant protein intracellularly. The st II signal sequence bears no similarity to the st I signal sequence and therefore it is considered a different structure.
In this patent application the inventors have taken advantage of both the pre and the pro region of STI to create a recombinant vehicle in which the nucleotide sequence coding for a peptide is fused inframe at the terminus of the pro region. Expression of the whole gene, resulted in the secretion of the recombinant peptide extracellularly and it was correctly processed. A schematic diagram of the principle is shown below: ##STR1## The advantage of this system is that the purification of the recombinant product becomes much simpler as the cells harboring such a recombinant vector can be grown in a synthetic medium and therefore the secreted peptide constitutes the major peptide present in the culture supernatant. A general method of purification of such a recombinant heterologous peptide (AngiotensinI) is described here.